Abstract
Purpose :
Dry eye results in pathological changes in the cornea including a disrupted epithelial barrier, loss of corneal epithelial progenitor cell identity and altered cell differentiation. In this study we investigate epithelial cell types and how their identities change with disease using a mouse model of autoimmune-mediated dry eye (Aire KO).
Methods :
Aire KO and wild type (WT) corneas were subjected to single nuclei (sn)RNA-seq (5 mice/group) using a 10X genomics platform. Epithelial subpopulations, potential lineage trajectories and signaling pathways involved with disease were identified using Seurat, Slingshot and Cellchat, respectively. snRNAseq outcomes were then validated through a combination of in situ hybridization/immunofluorescence and high-resolution confocal imaging.
Results :
snRNAseq analysis identified classical epithelial subtypes i.e., basal, wing and squamous cells, and showed these can be further sub-clustered into 3 basal, 2 limbal,1 wing, and 2 squamous clusters. Interestingly, the Aire KO cornea exhibited significant alterations in these sub-populations as well as the emergence of two new cell clusters. Additionally, lineage trajectory analysis predicted an unconventional differentiation pathway showing basal cells transitioning to either wing OR squamous cells through the 2 new clusters. Furthermore, Aire KO basal progenitors exhibited alterations in cell-cell communication including increased EGF and TNF signaling, and reduced PTN signaling. Immunofluorescent analysis of the Aire KO also identified an extensive increase in mTORC1 and mTORC2 signaling, suggestive of chronic wound healing.
Conclusions :
We identified novel homeostatic corneal epithelial subtypes and a striking alteration in epithelial cell differentiation in the Aire KO that may be mediated by specific alterations in basal cell signaling.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.